Understanding The Roles Of Mycobacteria Redox-related Proteins Hemerythrin-like Protein MSMEG₃312 And Universal Stress Protein Rv1996 In Drug Resistance

Mycobacterium tuberculosis is an ancient pathogen that causes tuberculosis and it claims 2 millions death each year.The prevalence of multiple drug-resistant TB and the extensively drug-resistant TB worsen the threat of human health.To understand the drug resistance mechanisms would lead to novel strategies to develop new therapies against M.tuberculosis.In this study,we explored the possible functions of mycobacteria redox-related proteins in drug resistance.MSMEG₃312 was predicted as a hemerythrin-like protein,which can carry oxygen and reversibly bind to oxygen,thus we reasoned that it should play important roles in the redox balance in M.smegmatis.So we constructed the msmeg₃312 knockout strain to compare the susceptibility to various drugs to its parent strain,me2155.The msmeg₃312 knockout strain showed increased erythromycin resistance.And the complementary strain partly lost this phenotype.To confirm the results,we compared the growth curves in liquid medium and its drug resistance on plate.Interestingly,we found the drug resistance was only limited to erythromycin,which its mechanism of action is by binding to the 50S subunit of the bacteria ribosomal complex and then inhibiting protein synthesis.However,there were no different MICs（minimum inhibitory concentration）of other antibiotics targeted on ribosome with different mechanisms,such as tetracyclines,aminoglycosides and chloramphenicol.In addition,we also evaluated the promoter inducement when treatment of erythromycin and it turned out erythromycin exerted great influence on expression of msmeg₃312 promoter.This is the first time to discover that hemerythrin-like protein MSMEG₃312 relates to drug resistance.According to these experiments we established correlative methodology.Meanwhile,compared to wild type BCG,M.bovis BCG with Rv1996 overexpression increased INH-susceptibility and H2O2-resistance,which indicates that Rv1996 is related to regulation of ROS in vivo.On the basis of experiment operating in M.smegmatis,we conducted the following assays.Proteomic analysis indicated it was because overexpression of Rv1996 affected KatG in some way.Further study showed that overexpression of Rv1996 did not cause the katG mutation or expression of katG in mRNA level.In addtion,the native gel to test catalase and peroxidase activity of KatG and the NBT reduction assay showed overexpression of Rv1996 increases KatG activity in vivo.Our result suggested overexpression of M.tuberculosis USP Rv1996 increased KatG activity and the activated KatG decreased mycobacterial INH resistance.Our findings would lead to novel strategies to develop new therapies.